Exploring the requirements for the hydrophobic scaffold and polar amine in inhibitors of M2 from influenza A virus

Article abstract of DOI:10.1021/ml100297w

Inhibitors targeting the influenza A virus M2 (A/M2) proton channel have lost their effectiveness due to widespread resistance. As a first step in the development of new inhibitors that address this problem, we have screened several focused collections of small molecules using two-electrode voltage patch clamp assays (TEVC) on Xenopus laevis oocytes. Diverse head groups and scaffolds of A/M2 inhibitors have been explored. It has been found that not only amine but also hydroxyl, aminooxyl, guanidine, and amidine compounds are active against the A/M2 proton channel. Moreover, the channel is able to accommodate a wide range of structural variation in the apolar scaffold. This study offers information to guide the next generation of A/M2 proton channel inhibitor design.

Full text of DOI:10.1021/ml100297w

LETTER
pubs.acs.org/acsmedchemlett
Exploring the Requirements for the Hydrophobic Scaffold and Polar
Amine in Inhibitors of M2 from Influenza A Virus
Jun Wang,† Chunlong Ma,§ Victoria Balannik,§ Lawrence H. Pinto,§ Robert A. Lamb,||,^ and William
F. DeGrado*,†,‡
†Department of Chemistry and ‡Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania,
Philadelphia, Pennsylvania 19104-6059, United States
§Department of Neurobiology and Physiology, Department of Biochemistry, Molecular Biology and Cell Biology, and ^Howard
Hughes Medical Institute, Northwestern University, Evanston, Illinois 60208-3500, United States
S Supporting Information
b
ABSTRACT: Inhibitors targeting the influenza A virus M2 (A/M2) proton channel have
lost their effectiveness due to widespread resistance. As a first step in the development of
new inhibitors that address this problem, we have screened several focused collections of
small molecules using two-electrode voltage patch clamp assays (TEVC) on Xenopus laevis
oocytes. Diverse head groups and scaffolds of A/M2 inhibitors have been explored. It has
been found that not only amine but also hydroxyl, aminooxyl, guanidine, and amidine
compounds are active against the A/M2 proton channel. Moreover, the channel is able to
accommodate a wide range of structural variation in the apolar scaffold. This study offers
information to guide the next generation of A/M2 proton channel inhibitor design.
KEYWORDS: Influenza A/M2, A/M2 channel inhibitor, two-electrode voltage clamp assay (TEVC)
nfluenza A viruses present a severe human health threat due to
the ease of transmission via the upper respiratory system.
A/M2 inhibitors, which might similarly be modified to improve
potency toward drug-resistant mutants in subsequent studies.
Inhibitors tested in this study were either synthesized or pur-
chased through commercial vendors.
The inhibitors were tested via a two-electrode patch clamp
assay (TEVC) using Xenopus laevis frog oocytes microinjectected
with RNA expressing the A/M2 protein as in a previous report.16
The potency of the inhibitors was expressed as the percentage
inhibition of A/M2 current observed after 2 min of incubation
with 100 μM compounds, and IC50 values were collected for
selected potent compounds.
A common feature of all known M2 channel inhibitors is the
presence of a polar headgroup, generally a primary or secondary
amine, connected to a hydrophobic scaffold. Interestingly, the
crystal and solid state NMR structures of amantadine complexed
A/M2 did not identify any specific interactions between the
amino group and the protein;9,17 instead, our most recent high-
resolution X-ray structure showed a cluster of well-organized
water molecules that might form hydrogen bonds with His37.18
The water cluster is highly polarized to stabilize an incoming
hydronium ion, which might be mimicked by the ammonium
group of the inhibitor. This observation led us to ask whether the
primary amine of amantadine might be substituted by other polar
groups without significantly affecting the activity. To explore
whether the amine can be substituted by any other polar head
I
Influenza A virus constantly undergoes antigenic drift and
antigenic shift, which allow them to escape from existing
immunity.1 Currently, there are two classes of small molecule
anti-influenza virus drugs:2 zanamivir and oseltamivir, which
target neuraminidase, and A/M2 channel inhibitors, which
include amantadine and rimantadine. Widespread resistance to
both oseltamivir (the only orally bioavailable neuraminidase
inhibitor) as well as A/M2 inhibitors presents a major health risk.
The A/M2 protein forms a homotetrameric proton selective
channel3,4 that is essential for viral replication.5,6 Amantadine
targets the A/M2 channel by blocking its pore.7-9 The number
of drug-resistant variants of A/M2 is limited by the very
conserved nature of the binding site within the channel. V27A,
L26F, and S31N are the only widely occurring drug-resistant
variants seen in transmissible strains of the virus.10-12
Extensive medicinal chemistry efforts have been devoted to
identifying A/M2 channel inhibitors with higher potency and
reduced side effects.2 However, very few chemotypes other than
the adamantane scaffold have been studied for A/M2 channel
inhibition.2,13 One exception is a spiro-piperidine 1, which
emerged from our previous structure-activity relationship
(SAR) study of 2-[3-azaspiro(5,5)undecanol]-2-imidazoline
(BL-1743).14 Further structure modification yielded another
class of spiran amines 2 that are not only active against wide
type (WT) A/M2 but also A/M2-V27A mutant channel.15
Encouraged by these initial results, we decided to explore both
headgroup and scaffold diversity to develop new generations of
Received: December 13, 2010
Accepted: January 29, 2011
Published: February 08, 2011
r
2011 American Chemical Society
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Table 1. Activities of Adamantane Analogues with Diverse Polar Head Groups on WT A/M2 Inhibition
a NA, not available.
groups, we examined the following panel of compounds based on
an adamantane scaffold (Table 1).
of the terminal amine (Table 2). In comparison, both the
glutarimide 16 and the aminoglurarimide 17 were only weakly
active against A/M2, probably due to the introduction of
hydrogen bond acceptors and the strong decrease in basicity.
In conclusion, these results are consistent with previous studies
showing that alcohols can substitute for amines in M2
inhibitors19 and expand the range of groups that can be con-
sidered for design.
Amantadine showed 91 ( 3% inhibition (IC50 = 16 μM)
against the WT A/M2 channel and was used as a benchmark for
comparison. The negatively charged 1-adamantyl sulfonic acid 3
was inactive, consistent with M2's function of stabilizing a
positively charged permeant ion. Amino-oxyl 4 and 1-adamantol
5 were nearly as active as the corresponding amine, giving a
similar degree of inhibition at 100 μM. Guanidine 6 (IC50 = 15
μM) had a similar activity to amantadine. In contrast, the
1-adamantyl biguanidine 8 was much less active than 1-adaman-
tyl guanidine 6, possibly due to the decrease of hydrophobicity or
the increased bulk of the polar group. Interestingly, when the
amine was substituted by amidine 7, the potency increased nearly
4-fold with an IC50 of 4 μM. N0-Hydroxyadamantane-1-carbox-
imidamide 9 was also less active than amantadine when compar-
ing percentage inhibition at 100 μM. Neutral compounds such as
amide 10 and 2-adamantanone 11 both showed decreased
activities relative to the corresponding amine. 2-Methyl-2-ada-
mantanol 12 (IC50 = 14 μM) had a higher activity than both
1-adamantol 5 (IC50 = 21 μM) and amantadine (IC50 = 16 μM).
This finding was consistent with earlier studies showing the in
vitro activity of 2-methyl-2-adamantol in influenza virus-induced
cytopathic effect (CPE) assay.19 2-Aminoadamantane 13 was
shown to have a similar activity as amantadine. In fact, a series of
2-substitited adamantane molecules are known to be highly
active against influenza A virus in in vitro viral CPE assay.20
Additionally, for the spiran class of inhibitors, converting
either the primary amine of 2 or the secondary amine of 1 to
the corresponding hydrazine in 14 and 15, respectively, de-
creased potency. This is possibly due to a decrease in the basicity
Pentacyclo[5.4.0.0.2,60.3,1005,9]undecane is a particularly good
synthetic precursor for multifunctional inhibitors, as the diketone
can be easily converted to a diol, hydroxyl amine, or hydroxyl
ketone.21 Our recent high-resolution X-ray crystal structure of A/
M2 revealed two layers of well-organized water molecules
positioned above the proton-accepting H37 residues.18 We
rationalized that an additional polar headgroup might potentially
hydrogen bond to or displace these water molecules.
The desired compounds were synthesized by standard proce-
dures from the starting diketone (Scheme 1). In this series,
monoamine 20 showed the most potent inhibition against the A/
M2 channel (IC50 = 8 μM), followed by amino alcohol 21 (IC50
= 24 μM). Diol 18 was also active, but the percentage inhibition
at 100 μM was smaller than that of the amino alcohol. Mono-
alcohol 19 showed only moderate inhibition against the A/M2
channel. The lower activities of compounds with an additional
polar headgroup might be due to the increased polarity, leading
to a greater dehydration penalty upon entering the A/M2
channel pore.
Encouraged by the result that diol compounds showed good
activity against A/M2, we next examined two commercially
available diols (22 and 23, stereochemistry is not defined by
the vendor). Both were found be less active than amantadine at
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Table 2. Activities of Spiran Amines/Hydrazines on WT A/M2 Inhibition
Scheme 1. Synthesis Scheme of A/M2 Inhibitors with More than One Polar Head Group
100 μM percentage inhibition. An additional hydroxyl group at
the 3-position of amantadine caused a dramatic decrease in A/
M2 inhibition (24). Another disubstituted oxyadamantane ana-
logue 25 was also not active. Taken together, this might be due to
either steric mismatch or decreased hydrophobicity. The ring-
contracted bisnoradamantane diol 26 was only moderately
active, highlighting that hydrophobic contacts are important
for tight binding.
the 2-position of rimantadine to connect the two adjacent helices
of A/M2, assuming the interfaces of the adjacent helices at
residue Asp44 are the pharmacologically relevant drug binding
sites; however, this inhibitor was found to be equally potent as
amantadine (IC50 = 16 μM) toward WT A/M2 but had no
inhibitory effect on either S31N or V27A mutants (Table 3).
Chen et al. reported a screen of 70 primary amines
including linear, aromatic, monocyclic, bicyclic, and tricyclic
compounds as A/M2 inhibitors.25 Five compounds were
found to have potency against A/M2 comparable to aman-
tadine. We have examined >30 additional amines with
various sized apolar groups, focusing primarily on com-
pounds with sizes and shapes roughly conforming to the
steric requirements of the binding site. Some of the struc-
tures are shown in Table 4. Consistent with previous find-
ings; amines with aromatic substitutions (28 and 29) are
significantly less active than the corresponding hydrocar-
bons, possibly due to the decreased hydrophobicity.
Earlier work by Clercq et al. examined additional 2-amino
substitution on the potency of rimantadine against influenza A
virus, finding two compounds with 1,2-diaminoethyl groups to
be just as potent as rimantadine.20,22 Taken together with our
data, we conclude that additional polar groups can be tolerated
but do not enhance the potency.
Compound 27 was designed by Chou et al.23 to potentially
target drug-resistant A/M2 mutants based on the solution NMR
structure of WT A/M2 (PDB code: 2RLF).24 The design
principle aimed to introduce an additional hydroxyl group at
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Table 3. Activities of A/M2 Inhibitor with More than One Polar Head Group on A/M2 Inhibition
a The percentage inhibition of 27 against the V27A and S31N mutants of A/M2 at a 100 μM concentration was 4 ( 1 and 15 ( 2%, respectively.
Table 4. Activities of A/M2 Inhibitor with Diverse Scaffolds on A/M2 Inhibition in TEVC
All linear alkyl amines tested (up to eight carbon atoms) in the
previous report were found to be inactive.25 Surprisingly, we
found the branched alkyl amine 30 to be nearly as active as
amantadine (IC50 = 21 μM), suggesting that the A/M2 channel
can accommodate a wide range of structural diversity.
A/M2 pore lining residues drug binding site V27, A30, and
G34 create a hydrophobic drug binding pocket that shows a
preference for inhibitors with clogP g 1.5.17 The results pre-
sented here, together with previous studies by Chen et al.25 show
that a minimum of eight aliphatic carbons are required for potent
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S31N mutants of A/M2 at a 100 μM concentration was less than
15 and 33%, respectively. (Amantadine showed 0 and 35%
inhibition against V27A and S31N A/M2 mutant at a 100 μM
concentration.) This is understandable, because both mutations
would increase the polarity of the pore.
A second mode of binding suggested by Chou and co-workers
involves a more peripheral site on the surface of the protein.24 Chou et
al. proposed that, if this mode of binding were correct, compound 27
should be a potent inhibitor of both WT protein as well as S31N,
given that the peripheral binding sites were on the surface of the
protein.23 On the other hand, the prediction of the pore-binding
model was that this compound should bind only to the WT protein.
We therefore tested this compound and found it to be a good
inhibitor of WT channels; however, it did not show significant
inhibition of V27A and S31N mutant forms. Thus, our results are
fully consistent with the pore-binding site being the pharmacologically
relevant site of inhibitor binding and do not support the peripheral
mode of binding. In summary, these results are fully consistent with
the expectation that drugs bind within the pore, and we will focus
future work on the design of new inhibitors onto this location.
Figure 1. Structure of amantadine (purple carbon atoms) in complex
with the transmembrane domain of M2, as determined by solid-state
NMR9 (PDB: 2KQT), with the water structure seen in the high-
resolution crystallographic structure (PDB: 3LBW) superimposed. Six
water molecules from 3LBW, shown as red spheres, lie above the four
His37 and Trp41 residues shown near the bottom of the structure (side
chains shown in stick). The apolar region of the drug projects into the
cavity formed by Val27, Ser31, and Ala30 (near the upper portion of the
structure), while the ammonium group projects downward toward the
water cluster. One helix has been removed for clarity.
’ ASSOCIATED CONTENT
S
Supporting Information. Experimental procedures for
b
the synthesis and characterization of A/M2 inhibitors (1H and
13C NMR, ESI-MS, and HRMS). This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
inhibition. Tetramethyl cyclopropane amine 31, which has seven
aliphatic carbons, showed weak inhibition (28% inhibition).
Compound 32 showed higher activity than amantadine and
has the same number of carbon atoms as amantadine (IC50 = 9
μM). Three bicyclic inhibitors (33, 34, and 35) were also tested
and found to be less active than amantadine at 100 μM
percentage inhibition. Four ring-expanded adamantane inhibi-
tors (36, 37, 38, and 39) showed similar activity as amantadine;
this result suggests that WT A/M2 is insensitive to minor scaffold
modifications.
Corresponding Author
*Tel: 215-898-4590. Fax: 215-573-7229. E-mail: wdegrado@-
mail.med.upenn.edu.
Funding Sources
This work was funded by the NIH (GM56423 and AI74571).
’ ACKNOWLEDGMENT
J.W. thanks Kathleen Molnar for help with HRMS data
collection.
As an initial step toward the design of inhibitors targeting
drug-resistant M2, it is important to first understand the mechan-
ism by which the WT protein binds inhibitors. X-ray crystal
structures17,18 and SSNMR structures9 of the channel have
indicated that amantadine binds with its aliphatic region project-
ing toward an apolar pocket (Figure 1). These structures indicate
that there is sufficient space in the pocket to accommodate larger
hydrophobic groups. The SAR shown in this paper for both the
polar headgroup and the apolar scaffold is consistent with such a
mode of binding. In the previous literature, almost all potent A/
M2 channel inhibitors are cyclic compounds with an amine
headgroup.2 In this study, we have found that (1) the amine
group is not essential for activity and can be substituted by
hydroxyl, guanidine, amidine, and aminooxyl groups; (2) potent
inhibition does not require a cyclic scaffold, so long as the shape
of the molecule conforms to the cavity, such as the linear
branched amine (30); (3) compound hydrophobicity is very
important for the potency of the inhibitor, and the effective A/
M2 inhibitor should possess clogP g 1.5 or 8 aliphatic carbon
atoms. While these compounds are active against the WT
protein, they were found to be less potent or inactive against
V27A and/or S31N mutant forms. The percentage inhibition of
all compounds other than amantadine against the V27A and
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